The composition and emission of volatile organic compounds in Eucalyptus spp. leaves. Anthony J. Winters Submitted in fulfillment of the requirements of the degree Doctor of Philosophy March, 2010 School of Biological, Earth and Environmental Sciences University of New South Wales PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: WINTERS First name: Anthony Other name/s: Jon Abbreviation for degree as given in the University calendar: School: Biological, Earth and Environmental Sciences Faculty: Science Title: The composition and emission of volatile organic compounds in Eucalyptus spp. leaves. Abstract 350 words maximum: (PLEASE TYPE) The emission of volatile organic compounds (VOCs) from plants has important effects on atmospheric chemistry and the carbon dynamics on plants. Eucalypts are amongst the highest emitters of VOCs, yet there are relatively few studies of emissions from this genus. The few studies published have examined emissions rates in relation to uncontrolled environmental variables. Only one study has examined these effects under controlled conditions. This thesis examined the factors that regulate VOC emissions from Eucalyptus globulus, one of the most widely cultivated eucalypt species. The emissions of isoprene, monoterpenes and the short-chained carbonyls formaldehyde, acetaldehyde and acetone were determined from four eucalypt species. Carbonyl emissions were generally comparable with rates reported for other species. There was large variation in diurnal isoprene and monoterpene emissions between species, but under standard conditions, isoprene emissions were much lower than previous reports. Monoterpenes were as much as six times greater than previous reports for some species. Emission of each carbonyl was correlated with its ambient concentration across different species. The effects of temperature and light on isoprene and monoterpene emissions from E. globulus were studied under laboratory conditions. Isoprene emissions exhibited the well described enzymatic response to increasing temperature, with a peak in emission at around 45 ƱC. Monoterpenes exhibited a large transient burst in response to incremental increases in leaf temperature. Analysis of the decay rate suggested a single pool contributed to the transient burst. I argue that emissions occur directly from oil glands, regulated only by a thin layer of cap cells above the gland. Light-dependent emission of cis-ocimene was also observed. Fumigation of leaves identified four compounds incorporated 13 C; isoprene (C5), cis-ocimene (C10), trans-caryophyllene (C15) and the tentatively identified C5 compound iso-valeraldehyde. Emissions were only detected in emitted compounds and no labelled uptake was identified in either oil glands or the remaining leaf tissue. The fraction of final 13C incorporation in the three terpenoids appeared to match the established paths of biosynthesis. Together, these data suggest that isoprene emissions from eucalypts may be a factor of two or more lower than previously estimated, while the case for monoterpene emissions remains unclear. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). …………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS Abstract The emission of volatile organic compounds (VOCs) from plants has important effects on atmospheric chemistry and the carbon dynamics on plants. They also constitute a fascinating evolutionary response to the many challenges faced by plants. Eucalypts are amongst the highest emitters of VOCs, yet there are relatively few studies of emissions from this genus. While known to emit isoprene and monoterpenes, the few published studies have examined emissions rates and composition in relation to uncontrolled environmental variables. Only one study has examined these effects under controlled conditions, but examined only three terpenoid compounds. This thesis examined the factors that regulate VOC emissions from Eucalyptus globulus, one of the most widely cultivated eucalypt species. The first major section of research examined emissions of isoprene, monoterpenes and the short-chained carbonyls formaldehyde, acetaldehyde and acetone from four species (Eucalyptus camaldulensis, Eucalyptus globulus, Eucalyptus grandis, and Eucalytpus viminalis) in south-western Australia. A smaller comparative study of carbonyl emissions was conducted on E. camaldulensis in south-eastern Australia. Carbonyl emissions were generally comparable with rates reported for other species, with diurnal emissions peaking at about 4, 75 and 34 nmol m-2 min-1 for acetone, formaldehyde and acetaldehyde respectively. There was considerable variation in diurnal isoprene and monoterpene emissions between species, but under standard conditions, isoprene emissions were between c. 440 and 1400 ug C m-2 h-1 across all species. In E. globulus, the best studied species to date, these emission rates are two to eight times lower than previous reports. Conversely, standard emission rates of monoterpenes ranged between 360 – 3300 ug C m-2 h-1 which, where data exists, is as much as six times greater than i previous reports for some species. Emission of each carbonyl was correlated with its ambient concentration across different species, but more weakly related to temperature. Acetaldehyde emission in particular was significantly correlated with transpiration, but not with sap flow or with ethanol concentrations in xylem sap. This suggests fermentation within the leaf and stomatal conductance are primary controlling processes, since differences in acetaldehyde exchange velocities between sites suggest stomata may indeed exert long term emission regulation, in contrast to compounds for which no biological sink exists. The second major section examined the effects of temperature and light on isoprene and monoterpene emissions from E. globulus. Isoprene emissions exhibited the well described enzymatic response to increasing temperature, with a peak in emission at around 45 °C. Even at high temperatures, the loss of carbon as a result of isoprene emission did not exceed 3 %. Monoterpenes, on the other hand, exhibited a large transient burst in response to incremental increases in leaf temperature that was c. 2 – 16 times the steady-state emission rate. These bursts took one hour or more to decay to a steady state. Analysis of the decay rate suggested a single pool contributed to the transient burst. Using evidence from this study and previous published reports, I argue that emissions occur directly from oil glands, regulated only by a thin layer of cap cells above the gland. Evidence is also presented for the light-dependent emission of cis- ocimene. These data suggest emission estimates of eucalypts using conventional models may be in serious error. The final piece of research sought to identify the source of monoterpenes from within leaves of E. globulus. Fumigation of leaves identified four compounds incorporated 13 C; isoprene (C5), cis-ocimene (C10), trans-caryophyllene (C15) and the tentatively identified C5 compound iso-valeraldehyde. Importantly, emissions were only detected ii in emitted compounds and no labelled uptake was identified in either oil glands or the remaining leaf tissue. The fraction of final 13C incorporation in the three terpenoids appeared to match the established paths of biosynthesis. The uptake of 13C incorporation agreed with monoterpene synthase activity studied in leaves of the same age. Confirmation and further elucidation of iso-valeraldehyde emission is required. The identification of 13C uptake by a single monoterpene and sesquiterpene is discussed against the dynamics of emission over the age of a eucalypt leaf. Together, these data suggest that isoprene emissions from eucalypts may be a factor of two or more lower than previously estimated, while the case for monoterpene emissions remains unclear. Estimates based on temperature-correcting models may be adequate under temperatures between 35 and 40 °C, but subject to more uncertainty below and above these temperatures. Elucidation of those mechanisms responsible for the novel behaviour reported here, and further model development, will go some way to resolving this uncertainty. iii ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another